Abstract

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

Many of the adaptive responses to N starvation are regulated at the level of gene expression, and some molecular components involved in that regulation have been described. Arabidopsis miR169 inhibits some aspects of the N starvation response, including NRT2.1 expression and N content (Zhao et al., 2011). CBL7 plays a positive role in the regulation of NRT2.4 and NRT2.5, root architecture, and root nitrate content (Ma et al., 2015). Although members of the NIN-LIKE PROTEIN (NLP) family are master regulators of the primary nitrate response in Arabidopsis (Konishi and Yanagisawa, 2013; Marchive et al., 2013; Liu et al., 2017), they have been implicated in the regulation of some elements of N starvation-inducible gene expression (Castaings et al., 2009). Rubin et al. (2009) reported that LATERAL ORGAN BOUNDARY DOMAIN family transcription factors (LBD37-39) are involved in the repression of a number of N starvation-inducible genes, as well as anthocyanin accumulation. Despite these advances, the molecular mechanisms underlying transcriptional regulation of N starvation responses remain largely unknown.

Using yeast one-hybrid (Y1H) screening, we identified transcriptional regulators of Arabidopsis NRT2.4, a N starvation marker gene (Kiba et al., 2012). These regulators are named NIGT1/HRS1s because they are highly homologous to a rice (Oryza sativa) protein known as NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) (Sawaki et al., 2013) and a member of them has already been named HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 (HRS1) (Liu et al., 2009). They belong to a small clade in the GARP-type transcription factor family, which are transcription factors widely distributed in eukaryotic organisms (Safi et al., 2017) and are expressed when N availability is high. They appear to act as direct repressors of NRT2.4 and possibly as direct repressors of an array of N starvation-regulated genes in order to repress N starvation responses under high N availability conditions. Our results also suggest that they indirectly sensitize phosphate (Pi) starvation responses such as activation of Pi starvation-induced gene expression (Puga et al., 2017) by directly repressing the repressors of the Pi starvation response. Together, these results define Arabidopsis NIGT1/HRS1s as important regulators of N starvation responses, with activity as balancers of N and Pi starvation responses.

RESULTS

Identification of Arabidopsis NIGT1/HRS1/HHO Family Members as NRT2.4 Promoter-Interacting Proteins

The full-length NRT2.4 promoter, extending 1886 bp upstream of the inferred initiation codon (Pro1886), is activated under N starvation and reports N availability (Kiba et al., 2012). To identify the regulatory factors of this promoter, we first defined the minimal NRT2.4 promoter region required for the N availability response by fusing a 5′-deletion series of the NRT2.4 promoter with sequences encoding GFP (Figure 1A). Stable Arabidopsis transformants harboring the Pro1886:GFP (P1886G), P639G, and P389G constructs had high levels of GFP fluorescence and transcript levels under N starvation conditions, whereas P254G did not respond to N availability (Figures 1A to 1C). This indicates that P389 includes the minimal sequence required for N availability-responsive expression.

Deletion Analysis of the Arabidopsis NRT2.4 Promoter and Isolation of NIGT1/HRS1/HHO Protein Family Members That Can Interact with the Deleted Promoter.

(A) Schematic of the full length and three truncated ProNRT2.4:GFP fusion constructs. P1886G represents the full-length 5′ upstream NRT2.4 promoter (1886 bp) fused with GFP. Deletion constructs are named for the length of the remaining promoter region.

(B) and (C) GFP florescence (B) and expression (C) in transgenic seedlings harboring the full-length or one of the three truncated NRT2.4 promoter:GFP constructs. Two independent lines (L1 and L2) of 7-d-old transgenic seedlings were grown for 3 d on MGRL agar plates with 10 mM nitrate (+N) or without an added nitrogen source (−N).

(C)GFP transcript levels were measured by qRT-PCR and normalized using the stably expressed At4g34270 as an internal control. Error bars represent sd (n = 4 independent pools of more than 10 whole plants).

(D) Y1H analysis of interactions between NIGT1 subfamily members and the NRT2.4 promoter. The NRT2.4 P389 promoter was used for bait. NIGT1.1, NIGT1.2, and NIGT1.3 cDNAs were cloned into pGAD424 as prey. Transformed yeast strains were grown either on permissive medium (without aureobasidin A [−AbA]) or on selective medium (with aureobasidin A [+AbA]).

(E) Interactions between NIGT1/HRS1/HHO family members and the NRT2.4 promoter in transient assays. Transient assays were conducted using constructs harboring the NRT2.4 P389 promoter fused with the luciferase gene (P389:FLUC) as a reporter. Constructs expressing GFP or NIGT1-related transcription factors fused with two stringent transcriptional activation domains VP16 (VP) were used as effectors. Promoter activities are given as FLUC/RLUC activity ratios. A vector with the CaMV 35S promoter fused to the RLUC reporter was used as an internal control for normalization of bombardment efficiency. Error bars represent sd (n = 4 independent pools of more than 8 seedlings). Asterisks indicate statistically significant differences (P < 0.05; Student’s t test). Each experiment was conducted twice with similar results.

The P389 fragment was used as bait in a Y1H screening experiment to identify factors that regulate this promoter (Figure 1D). The bait-reporter yeast strain, which harbors a construct consisting of three tandem copies of P389 fused to the aureobasidin A-resistance gene, was transformed with a prey library composed of 1498 Arabidopsis transcription factor cDNAs (Mitsuda et al., 2010). We sequenced prey plasmids from the 27 aureobasidin A-resistant colonies and found that 21 of them were GARP (GOLDEN2, ARR-B, Psr1) GOLDEN2-like (GARP-G2) family members AT1G25550, AT1G68670, and AT3G25790 (Figure 1D; Supplemental Table 1). Among 40 GARP-G2 family transcription factors in Arabidopsis, these three proteins fall into a subclade consisting of four members within a larger clade consisting of seven members (Supplemental Figure 1). Since the subclade members are highly homologous to a rice protein known as NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) (Sawaki et al., 2013), we designate these proteins collectively as Arabidopsis NIGT1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 (NIGT1) subfamily members and individually as NIGT1.1/HRS1 HOMOLOG3 (NIGT1.1), NIGT1.2, NIGT1.3, and NIGT1.4. The other members of the larger clade have already been named HRS1 HOMOLOG4 (HHO4) to HHO6 (Liu et al., 2009; Medici et al., 2015). Thus, the larger clade with seven members was designated the NIGT1/HRS1/HHO family.

To further test the interactions between NIGT1s and the NRT2.4 promoter, we conducted particle bombardment-mediated transient assays in Arabidopsis seedlings (Figure 1E). Bombardment with an effector plasmid expressing each of the NIGT1s fused with two stringent transcriptional activation domains VP16 (VP) increased firefly luciferase (FLUC) activity under control of the NRT2.4 P389 promoter. HHO4-VP, HHO5-VP, or HHO6-VP fusion proteins also activated the promoter, but neither GFP nor GARP-G2 family members in other clades (e.g., PHR1 and AT3G10760) did. This indicates that, although there are differences in levels of activation, each of the NIGT1/HRS1/HHO family members interacts with the NRT2.4 promoter. In this study, we focused on the characterization of four highly homologous members NIGT1.1 through 1.4.

Arabidopsis NIGT1 Subfamily Members Are Transcriptional Repressors That Bind to the NRT2.4 Promoter

To determine the molecular function of the NIGT1s, we confirmed that these proteins are localized in the nuclei of transgenic plants expressing NIGT1.1-GFP or NIGT1.2-GFP fusions under the control of the CaMV 35S promoter (Figure 2) (Medici et al., 2015; Nagarajan et al., 2016). The localization pattern was not altered by N availability (Figure 2A).

Arabidopsis NIGT1 Subfamily Members Are Nuclear-Localized Transcriptional Repressors and Interact with the NRT2.4 Promoter in a Sequence-Specific Manner.

(A) Localization of GFP in roots of 5-d-old transgenic seedlings expressing NIGT1.1-GFP (NIGT1.1-GFP) or NIGT1.2-GFP (NIGT1.2-GFP) under the control of the CaMV 35S promoter grown on 0.5× MS agar plates with (+N) or without a nitrogen source (−N). BF/GFP represents merged images of bright field and GFP. Bar = 25 μm.

(B) The effect of NIGT1 subfamily members on GAL4 promoter activity in transient assays. GAL4 promoter activity from ProGAL4:FLUC was measured when cobombarded with the GAL4 DNA binding domain (GDB) fused to GFP or NIGT1.1-1.4. Promoter activities are given as FLUC/RLUC activity ratios. A vector containing RLUC driven by the CaMV 35S promoter (ProCAMV35S:RLUC) was used as an internal control for normalization of bombardment efficiency.

(C) to (G) Interactions between NIGT1 subfamily members and mutated NRT2.4 promoters in transient assays.

(D) to (G) Transient assays were conducted using constructs harboring the promoters illustrated in (C) fused with FLUC or FLUC only (Empty) as reporters. As effectors, constructs expressing NIGT1.1 (D), NIGT1.2 (E), NIGT1.3 (F), and NIGT1.4 (G) fused with two VP16 domains (VP) were used. ProCAMV35S:RLUC was used as an internal control for normalization of bombardment efficiency. The promoter activities are shown as FLUC/RLUC activity ratios. B1m and B2m represent mutated NRT2.4 promoters at the B1 and B2 motifs, respectively. Different lowercase letters indicate statistically significant differences as indicated by Tukey’s HSD test (P < 0.05). Error bars represent sd of four ([B], [D], and [E]) and three ([F] and [G]) independent pools of more than eight seedlings. Each experiment was conducted twice with similar results.

To test whether NIGT1s have transcriptional activator or repressor activity, transient assays were conducted using a reporter plasmid harboring a GAL4 promoter:FLUC (ProGAL4:FLUC) fusion and an effector plasmid expressing NIGT1 fused with a GAL4 DNA binding domain at its N terminus (GAL4DB-NIGT1.1 through GAL4DB-NIGT1.4). Expression of GAL4DB-NIGT1s reduced GAL4 promoter activity compared with that of control (GAL4DB-GFP), indicating that NIGT1s act as transcriptional repressors (Figure 2B).

In vitro DNA binding site selection experiments and electrophoretic mobility shift assays had previously identified rice NIGT1 binding sequences as GAATATTC and GAATC (Sawaki et al., 2013). Recently, DNA affinity purification sequencing (DAP-seq) indicated that the binding motifs of Arabidopsis NIGT1.1, NIGT1.2, and NIGT1.4 are identical to those of rice NIGT1 (O’Malley et al., 2016). There is one GAATATTC (B1) and three GAATC (B2) motifs within the Arabidopsis NRT2.4 P389 promoter (Supplemental Figure 2). To determine whether the NIGT1 proteins bind to P389 at these motifs, we tested the interactions between mutant promoters and NIGT1s in transient assays in Arabidopsis seedlings (Figures 2C to 2G). Reporter plasmids harboring mutant NRT2.4 promoters that were mutated (as shown in Figure 2C) in either the B1 motif (B1m), the B2 motifs (B2m), or in both the B1 and B2 motifs (B1mB2m) were fused with FLUC, and effector plasmids expressing NIGT1.1-VP to NIGT1.4-VP were used to assay activity. Although mutations in either the B1 or B2 motifs resulted in significantly decreased FLUC expression, the B1mB2m double mutation resulted in complete loss of interaction (Figures 2D to 2G), indicating that full NIGT1 binding within the NRT2.4 promoter requires both motifs (NIGT1 binding motifs).

To confirm the interactions in vivo, transgenic plants constitutively overexpressing NIGT1.1-GFP (NIGT1.1ox), NIGT1.2-GFP (NIGT1.2ox) fusion genes, or GFP (GFPox) under control of the CaMV 35S promoter were used in chromatin immunoprecipitation (ChIP)-quantitative reverse transcription-PCR (qPCR) assays with an anti-GFP antibody. We analyzed two regions of the NRT2.4 promoter having multiple NIGT1 binding motifs (#2 and #3) and three regions without the motif (#1, #4, and #5) (Figure 3). In addition, promoter regions of previously reported qPCR reference genes At2g28390 (#6) and At4g34270 (#7) (Dekkers et al., 2012) were chosen as controls (Figure 3A). Regions #2 and #3 were enriched in NIGT1.1ox and NIGT1.2ox compared with GFPox, but no significant enrichment was found on any other regions tested (Figure 3B). When transgenic lines expressing NIGT1.3-FLAG (eNIGT1.3ox), NIGT1.4-FLAG (eNIGT1.4ox), or GFP-FLAG (eGFPox) fusion genes under control of the β-estradiol-inducible promoter were analyzed using ChIP-qPCR and an anti-FLAG antibody, we found enrichment of the same regions (#2 and #3) (Figure 3B). These results show that all NIGT1 subfamily members associate with NRT2.4 promoter in vivo.

Arabidopsis NIGT1 Subfamily Members Are Associated with the NRT2.4 Promoter.

NIGT1 interaction with the NRT2.4 promoter was analyzed using ChIP assays.

(A) Diagrams of NRT2.4 and control (At2g28390 and At4g34270) loci and the locations of B1 (GAATATTC, red line) and B2 (GAATC, blue lines) motifs. Black bars and numbers 1 to 7 indicate DNA regions used in the ChIP assay.

(B) ChIP-qPCR assays were performed using 10-d-old transgenic seedlings. Left graph: Transgenic lines expressing GFP (GFPox), NIGT1.1-GFP (NIGT1.1ox), or NIGT1.2-GFP (NIGT1.2ox) fusion genes under control of the CaMV 35S promoter were used and immunoprecipitated with an anti-GFP antibody. Right graph: Transgenic lines expressing GFP-FLAG (eGFPox), NIGT1.3-FLAG (eNIGT1.3ox), or NIGT1.4-FLAG (eNIGT1.4ox) fusion genes under control of the β-estradiol-inducible promoter treated with 10 μM β-estradiol for 24 h were used and immunoprecipitated with an anti-FLAG antibody. The percentages of the DNA coimmunoprecipitated with antibody relative to input DNA are indicated. Numbers on the x axis indicate DNA regions shown in (A). Error bars represent sd (n = 3 independent pools of more than 10 whole plants). Asterisks indicate statistically significant differences (*P < 0.01, Student’s t test). Each experiment was conducted twice with similar results.

Arabidopsis NIGT1 Subfamily Members Repress NRT2.4 Expression in Vivo

To investigate the biological significance of interactions between the NRT2.4 promoter and NIGT1 proteins, β-estradiol (Est)-inducible overexpression lines were generated (eNIGT1.1ox to eNIGT1.4ox), and expression was measured with quantitative real-time RT-PCR (qRT-PCR) after Est treatment (Figure 4). Each of the Est-inducible lines overexpresses the corresponding NIGT1s in response to exogenous Est treatment. Compared with the vector control line eVC, inducible overexpression of the NIGT1s reduced N starvation-inducible NRT2.4 expression (Figure 4A). A similar reduction in NRT2.4 expression was observed in constitutive overexpressors NIGT1.1ox and NIGT1.2ox (Supplemental Figure 3A). To examine whether the reduction occurs at the level of transcription, we introduced the P1886G reporter into eNIGT1.2ox by crossing (P1886G x eNIGT1.2ox) and observed that GFP fluorescence was completely absent from roots after Est treatment (Figure 4B). qRT-PCR assays confirmed that GFP transcription is reduced by NIGT1.2 overexpression in a manner similar to that of endogenous NRT2.4 (Supplemental Figure 3B). These results suggest that NIGT1s repress NRT2.4 promoter expression by directly binding to it in vivo.

We isolated homozygous T-DNA insertion mutants in order to examine the effect of the loss of NIGT1 function on NRT2.4 expression (Supplemental Figure 4A). We tested to see if the homologous T-DNA insertions resulted in null mutants by RT-PCR. No full-length transcripts of corresponding NIGT1s were amplified by RT-PCR with saturating cycles in any single mutant (Supplemental Figure 4B), indicating that the T-DNA insertions resulted in null alleles. A series of double, triple, and quadruple mutants were produced by crossing the T-DNA mutants, and these mutants were analyzed using NRT2.4 expression assays (Figures 4C and 4D). There were no significant differences in NRT2.4 expression relative to wild-type levels in any of the single mutants, double mutants nigt1.1 nigt1.2, or nigt1.3 nigt1.4, or in any combination of triple mutants grown in half-strength Murashige and Skoog plates (0.5× MS containing ∼19.7 mM nitrate and 10.3 mM ammonium). However, expression was significantly upregulated (or derepressed) in all quadruple mutant (nigtQ) combinations (Figure 4C) unless plants were under N starvation conditions (Figure 4D).

We have confirmed that introduction of a genomic fragment containing either NIGT1.1 or NIGT1.2 complements the NRT2.4 expression phenotype in nigtQ (Supplemental Figure 5A). Collectively, these results indicate that NIGT1 proteins act redundantly to directly repress NRT2.4 expression under high N availability.

Expression of Arabidopsis NIGT1 Subfamily Genes Is Regulated by Nitrogen and Nitrate Availability

Because deregulation of NRT2.4 in nigtQ is N dependent (Figure 4D), we grew wild-type seedlings under different N conditions. Seedlings were incubated on agar plates containing 5 mM ammonium nitrate (NH4NO3) or without a nitrogen source for 3 d, and expression levels were assayed in shoots and roots by qRT-PCR (Figure 5). NIGT1.1 and NIGT1.2 were expressed in shoots and roots, and NIGT1.3 and NIGT1.4 were detected only in roots of NH4NO3-grown seedlings (Figure 5A). Under N starvation conditions, transcript levels of all NIGT1s except NIGT1.3 were significantly downregulated compared with NH4NO3-grown seedlings, and we observed no alteration of expression patterns in shoots or roots (Figure 5A). We then tested the effect of other N sources on NIGT1 expression in seedlings incubated with nitrate, ammonium, ammonium nitrate, Gln, urea, or without any added nitrogen (Figure 5B). Each of the nitrogen sources resulted in higher transcription levels of NIGT1.1 and NIGT1.2 than under N starvation conditions (Figure 5B), indicating that NIGT1.1 and NIGT1.2 expression is positively regulated by N availability. The expression levels of NIGT1.4 were higher when incubated with nitrate and ammonium nitrate but were lower with ammonium, Gln, and urea (reduced N metabolites) than under N starvation conditions. Compared with ammonium nitrate, nitrate induced the expression of NIGT1.3, whereas reduced N metabolites repressed it (Figure 5B). This observation agrees with a previous report indicating that NIGT1.3 and NIGT1.4 are nitrate-inducible (Medici et al., 2015). Given that the expression levels of NIGT1.3 and NIGT1.4 under N starvation conditions were higher than when grown with reduced N metabolites, it is likely that the expression of NIGT1.3 and NIGT1.4 is regulated by the balance between nitrate induction, repression, and derepression in response to reduced N metabolite availability, as has been reported for many nitrate-inducible genes, including NRT2.1 (Gansel et al., 2001; Nazoa et al., 2003; Widiez et al., 2011; Camañes et al., 2012).

(D) Expression levels of NIGT1s and NRT2.4 in roots in response to nitrogen supplement. Ten-day-old Col-0 seedlings N starved for 3 d were transferred to NH4NO3 plates or exposed to N starvation conditions and harvested for the indicated times. Error bars represent sd (n = 3 independent pools of more than 10 roots). Expression levels were analyzed by qRT-PCR and normalized using At4g34270 as an internal control. Each experiment was conducted twice with similar results.

In order to compare the patterns of NIGT1s and NRT2.4 expression in detail, we followed time-course expression patterns of them during N deprivation in roots. Seedlings grown in 0.5× MS were transferred to N-free media and expression was measured at daily intervals over the course of 5 d. Transcript levels of NIGT1.1, NIGT1.2, and NIGT1.4 gradually decreased after transfer, but NRT2.4 steadily increased (Figure 5C). Although there was no significant decrease in NIGT1.3 transcript levels when measured at daily intervals (Figure 5C), there was a transient decrease when measured at shorter intervals (Supplemental Figure 6A). This is consistent with a previous report that NIGT1.3 is an early nitrogen deprivation-downregulated gene (Menz et al., 2016).

We further analyzed the spatial expression patterns of NIGT1s in transgenic seedlings harboring a fusion between the NIGT1 promoter and the GUS reporter gene (Figure 6). GUS staining patterns in organs in response to N availability were consistent with our qRT-PCR results (compare Figures 5A and 6). In seedlings grown under high N availability, promoter activity of NIGT1s was observed not only in root epidermal cells, where NRT2.4 is expressed (Kiba et al., 2012), but also in many other cells and tissues. NIGT1.1 and NIGT1.2 expression was detected in most tissues, with hypocotyl and root cap being the exceptions. NIGT1.3 and NIGT1.4 appeared to be expressed in most cell types in the older part of the root, but not in the root tip region (Figure 6).

Identification of NIGT1 Target Genes

NIGT1s are expressed much more broadly than NRT2.4 (Figure 6), suggesting that there are other genes directly repressed by NIGT1s than NRT2.4. To identify NIGT1 direct target genes, we conducted combinatorial microarray analyses to allow a genome-wide search for genes bound and repressed by NIGT1s. We generated transgenic lines that overexpress NIGT1.1-VP or NIGT1.2-VP in an Est-inducible manner in the wild-type Col-0 background (pER-NIGT1.1-VP or pER-NIGT1.2-VP). These lines show upregulation of NRT2.4 with Est treatment under high N availability conditions (Supplemental Figure 7), suggesting that other NIGT1-bound genes would also be induced in these lines. Using the pER-NIGT1.2-VP and NIGT1.2ox transgenic plants, two sets of microarray data were produced. The first data set was obtained from pER-NIGT1.2-VP seedlings incubated with or without 10 μM Est for 24 h under high N availability. These seedlings had significantly upregulated probes in response to Est treatment and were recovered as NIGT1-bound genes (Welch’s t test, P < 0.05; Benjamini-Hochberg procedure, false discovery rate [FDR] < 0.1; fold change [FC] > 0). NIGT1.2ox and wild-type seedlings were incubated under N starvation conditions for 3 d. These seedlings had significantly downregulated probes in NIGT1.2ox and were recovered as NIGT1-repressed genes (P < 0.05, FDR < 0.1, FC < 0). Shoots and roots were analyzed separately. We expect that the genes delineated by combining these data are enriched in NIGT1 direct targets (Figure 7).

In roots, 2390 probes were identified as upregulated in pER-NIGT1.2-VP with Est treatment and 1353 were downregulated in NIGT1.2ox. A comparison between these two probe lists resulted in 419 putative direct targets in roots (Figure 7A; Supplemental Data Sets 1 to 3). We identified 2807 probes that were upregulated in Est-treated pER-NIGT1.2-VP shoots and 2172 that were downregulated in NIGT1.2ox shoots, yielding 310 putative direct targets (Figure 7B; Supplemental Data Sets 4 to 6). To validate this search procedure, seven genes (GLN1;4, NRT2.5, GDH3, DUR3, AMT1;1, NLP3, and SPX1) were arbitrarily selected, and their expression was analyzed in NIGT1.1ox, NIGT1.2ox, and nigtQ plants (Figure 8). All of these genes were repressed in NIGT1 overexpression lines under N starvation conditions but were derepressed in nigtQ under high N availability (Figures 8A and 8B). Introduction of a genomic fragment containing either NIGT1.1 or NIGT1.2 into nigtQ complemented the derepression of these genes (Supplemental Figure 5B). Furthermore, ChIP-qPCR experiments confirmed the association of NIGT1 with the NRT2.5, GLN1;4, and GDH3 promoters at the NIGT1 binding motifs (Figures 8C and 8D). Using different methods, Safi et al. (2018) recently reported that NRT2.4, NRT2.5, and GDH3 are directly repressed by NIGT1s. These results strongly support the validity of our lists as enriched for NIGT1 direct targets.

We also found N starvation downregulated genes, or N-inducible genes in the lists. They include well-known nitrate-inducible genes such as NRT1.1/NPF6.3, LBD37, and CIPK23 (Ho et al., 2009; Rubin et al., 2009), as well as important genes in the regulation of Pi-starvation responses such as class I SYG1/Pho81/XPR1 domain protein (SPX) genes, SPX1, SPX2, and SPX4, and PHOSPHATE2 (PHO2) (Liu et al., 2012; Huang et al., 2013; Puga et al., 2014) (Supplemental Data Sets 3 and 6). These results suggest that NIGT1 proteins have regulatory functions other than repressing N starvation-inducible genes.

Furthermore, all NIGT1s except for the overexpressed NIGT1.2 were found in the direct target list (Supplemental Data Sets 3 and 6). Overexpression of NIGT1.1 or NIGT1.2 represses endogenous NIGT1s (Figure 9A). ChIP-qPCR experiments confirmed NIGT1 association with all NIGT1 promoters around the NIGT1 binding motifs (Figures 9B and 9C), indicating that an auto-negative feedback-type regulation system, as has been reported for NIGT1 in rice (Sawaki et al., 2013), is also active in Arabidopsis.

Because nigtQ mutants were indistinguishable from the wild type in our high N availability/N-starved conditions (Supplemental Figure 9), we measured N-related physiological parameters in both NIGT1 overexpressors and nigtQ mutants (Figure 10; Supplemental Figure 10). NIGT1 overexpressors were analyzed under N starvation conditions, when most NIGT1 expression is downregulated in wild-type seedlings. The nigtQ mutant line was compared with the wild type under high N availability, when most NIGT1 expression is high in wild-type seedlings (Figure 5). N content was lower in NIGT1.1ox and NIGT1.2ox plants than in the GFPox line (Figure 10A). On the other hand, nigtQ had increased N content in shoots and roots compared with the wild type (Figure 10B). NIGT1.1ox and NIGT1.2ox contained less nitrate (Figure 10C). Although the nigtQ line had more nitrate than control seedlings (Figure 10D), there was no significant difference in soluble protein, chlorophyll, or total amino acid contents between nigtQ lines and wild-type seedlings (Supplemental Figures 10A to 10C). Because levels of glutamine and asparagine were higher but histidine, proline, and valine levels were reduced, the total amino acid levels were not significantly altered in the mutant (Supplemental Figures 10C and 10D).

(C) and (D) Nitrate contents in shoots and roots of NIGT1 overexpressors grown under N starvation (C) and nigtQ grown under high N availability (D). Error bars represent sd of three (C) and five (D) independent pools of the indicated tissue from more than 10 plants.

(E) and (F) Nitrate influx in NIGT1 overexpressors grown under N starvation (E) and nigtQ grown under high N availability (F). Influx was measured after short-term labeling with complete nutrient solution containing 15N-labeled nitrate at the indicated concentrations. LATS activity was calculated by subtracting the influx measured at 0.2 mM nitrate from the influx measured at 6 mM nitrate. Error bars represent sd of five (E) and four (F) independent pools of more than 10 whole plants.

(G) and (H) Expression levels of high-affinity nitrate transport-related genes NAR2.1/NRT3.1 (NAR2.1), NRT1.1/NPF6.3 (NRT1.1), and NRT2.1 in roots of NIGT1 overexpressors grown under N starvation conditions (G) and the nigt1.1 nigt1.2 nigt1.3 nigt1.4 (nigtQ) quadruple mutant grown under high N availability (H). Error bars represent sd of four (G) and five (H) in dependent pools of more than ten roots.

(A), (C), (E), and (G) Transgenic plants constitutively expressing NIGT1.1-GFP (NIGT1.1ox) or NIGT1.2-GFP (NIGT1.2ox) fusion genes or GFP (GFPox) under control of the CaMV 35S promoter grown for 7 d on 0.5× MS agar plates were transferred to and incubated on MGRL plates without N source for 3 d.

Because many genes involved in high-affinity nitrate uptake were on the root tissue direct target list (Supplemental Data Set 3), high-affinity nitrate influx was measured (Figures 10E and 10F). In N-starved NIGT1.1ox and NIGT1.2ox plants, influx was reduced in the high-affinity range at 0.02 and 0.2 mM (Figure 10E). In contrast, nigtQ plants grown under high N availability showed increased high-affinity influx at 0.2 mM, but no significant difference in low-affinity influx was detected (Figure 10F). Not surprisingly, alterations in high-affinity influx were positively correlated with the expression levels of high-affinity transport-related genes, NRT2.4, NRT2.5, NAR2.1/NRT3.1, and NRT1.1/NPF6.3 (Figures 8A, 8B, 10G, and 10H). Expression was repressed in NIGT1 overexpressors under N starvation conditions and derepressed in nigtQ under high N availability. We also tested the expression of the major high-affinity nitrate transporter gene NRT2.1, though it was not in our direct target list. NRT2.1 expression was positively correlated with high-affinity nitrate influx in a way similar to other direct target genes (Figures 10G and 10H). Nitrogen content, nitrate uptake, nitrate accumulation, and high-affinity nitrate transport-related gene expression phenotypes of nigtQ were complemented by introducing a genomic fragment containing NIGT1.2 (Supplemental Figures 5C to 5E).

Given that nigtQ plants had higher N uptake and N content (Figure 10) without any positive effect on growth under high N availability conditions (Supplemental Figure 9), it could be that the nigtQ line is impaired in its effective use of N under high N availability conditions. Collectively, these results suggest that NIGT1s suppress N starvation responses, especially responses that enhance N acquisition and N reserves, by directly repressing their target genes under high N availability (Figure 12A).

DISCUSSION

Direct Repression of Nitrogen Starvation-Inducible Genes by Arabidopsis NIGT1 Subfamily Members under High Nitrogen Availability

Although Arabidopsis LBD37, LBD38, and LBD39 (LBDs) are involved in the repression of a large number of N starvation-inducible genes, it is not known whether these genes are directly regulated by LBDs (Rubin et al., 2009). LBDs and NIGT1s are both regulated in response to N or a combination of N and nitrate availability, and expression of these genes is high under high N availability and low under low N availability (Figure 5) (Rubin et al., 2009). They also share many repression targets, including NRT1.1/NPF6.3, NRT2.1, NRT2.5, NAR2.1/NRT3.1, GDH3, GLN1;4, NLP5, and NAC55 (Supplemental Data Sets 3 and 6) (Rubin et al., 2009). The emerging model is that NIGT1 proteins and LBDs appear to act in the same pathway. Given that NIGT1 proteins directly repress the target genes, it can be hypothesized that LBDs act upstream of NIGT1s. However, microarray data published by Rubin et al. (2009) show that the expression of NIGT1s is not altered in the overexpressors and null mutants of LBDs, except that NIGT1.4 expression was lower in the LBD37 overexpressor under N starvation conditions (Rubin et al., 2009). This indicates that LBDs and NIGT1s do not function in the same pathway. Because our data indicate that LBD37 is a direct target of NIGT1s in roots (Supplemental Data Set 3), NIGT1s and LBDs likely function in different pathways but influence each other through negative regulatory interactions to repress overlapping sets of N starvation-inducible genes. This might explain why loss of NIGT1 activity did not result in full derepression of NIGT1 direct targets under high N availability conditions (Figures 4D and 8B). However, further experiments, such as characterization of mutants that have lost both LBD and NIGT1 functions, would be required to test this hypothesis.

NIGT1s Are Repressors of Nitrogen Starvation Responses in Arabidopsis

By combinatorial microarray analyses, we identified 419 root and 310 shoot direct target genes of NIGT1s (Figure 7; Supplemental Data Sets 3 and 6). Although direct targets were enriched with N starvation-inducible genes involved in various pathways, and N uptake and N content were significantly increased in nigtQ plants (Figure 10), we could not detect any alterations in growth or development of nigtQ (Supplemental Figure 9). A possible explanation could be that N starvation-inducible genes are redundantly repressed by NIGT1s and other factors, including LBDs (Rubin et al., 2009). Other members of the NIGT1/HRS1/HHO family could serve as redundant factors in this regulatory scheme because they also interact with the NRT2.4 promoter (Figure 1E). Alternatively, N starvation responses could also be regulated at the posttranscriptional level. Consistent with this idea, AMT1;1 and NRT2.1 are subjected to posttranscriptional regulation, a process that is dependent on N availability (Yuan et al., 2007b; Laugier et al., 2012), and many enzymes in primary metabolism are known to be regulated at the posttranslational level in response to environmental factors (MacKintosh, 1998). These observations suggest that N starvation responses are a critical biological process that is tightly controlled by multiple overlapping layers of regulation. In any case, we propose that the transcriptional repression of N starvation-inducible genes by NIGT1s is an important component of the N starvation response regulatory system (Figure 12A).

The Role of NIGT1s in Optimizing Nitrogen Acquisition and Utilization under Fluctuating Nutritional Conditions

We showed that the expression levels of NIGT1s decrease upon transfer to N starvation conditions, followed by a rapid increase after N and/or nitrate supplementation (Figures 5C and 5D; Supplemental Figure 6). Given that the NIGT1s are early nitrate-inducible genes and are directly regulated by NLPs and NRT1.1/NPF6.3 (Konishi and Yanagisawa, 2013; Marchive et al., 2013; Medici et al., 2015), the role of NIGT1s would likely be to repress N starvation-inducible genes immediately upon sensing the availability of nitrate. Because NIGT1.1 and NIGT1.2 expression is also induced by reduced N sources (Figure 5B), NIGT1.1 and NIGT1.2 should play a similar role upon detection of a reduced N source. Furthermore, data presented here show that nitrate-inducible genes are also repressed by NIGT1s (Figure 7D; Supplemental Data Sets 3 and 6), meaning that NIGT1s also participate in the feedback repression of nitrate-inducible genes. This sort of activity by NIGT1s would enable plants to swiftly respond to fluctuating N availability and demand in order to fine-tune N responses so that N acquisition and utilization can be optimized.

Several studies have implicated NIGT1s in the regulation of Pi responses, though no molecular mechanism has been pinpointed (Liu et al., 2009; Medici et al., 2015; Nagarajan et al., 2016). Liu et al. (2009) reported that NIGT1.4 (HRS1) expression is induced by Pi deficiency and that an NIGT1.4 overexpressor line is susceptible to Pi deficiency-elicited primary root growth repression and root hair differentiation enhancement. An overexpressor line and mutants of NIGT1.2 (HHO2) exhibit alterations in Pi starvation responsive genes and Pi content (Nagarajan et al., 2016). In our study, we identified three class I SPX and PHO2 genes as NIGT1 direct targets (Supplemental Data Sets 3 and 6), supporting the notion that NIGT1s are involved in the regulation of Pi responses. Since class I SPXs and PHO2 are known to be major negative regulators of Pi starvation responses (Liu et al., 2012; Huang et al., 2013; Puga et al., 2014), it is possible that NIGT1s sensitize Pi starvation responses indirectly through repression of the negative regulators (Figure 12B). As expected, NIGT1 overexpressor lines had higher levels of Pi content and Pi transporter expression (PHT1;1 and PHT1;4) than the wild type and the nigtQ mutant had lower levels than the wild type (Figures 11A to 11E). We also found that expression of each of the NIGT1s is induced by Pi starvation, but only under high N availability (Figure 11F), indicating that NIGT1 action in the regulation of Pi starvation responses is N dependent, as described by Medici et al. (2015) for NIGT1.3 (HHO1) and NIGT1.4 (HRS1) in primary root growth repression. Although further study will be required to fully understand the mechanism of Pi starvation response regulation by NIGT1s, we propose that NIGT1s have a dual function as direct repressors of N starvation responses and as indirect sensitizers of Pi starvation responses to balance N and Pi starvation responses (Figure 12B). Such a mechanism would enable plants to divert energy required for N starvation responses to Pi starvation responses under high N availability.

(A) and (B) Phosphate (P) content in shoots and roots of the NIGT1 overexpressor (A) and the nigt1.1 nigt1.2 nigt1.3 nigt1.4 quadruple (nigtQ) mutant (B). Fourteen-day-old 0.5× MS-grown seedlings were used in the assay.

(C) P content in wild-type (Col-0), the nigt1.1 nigt1.2 nigt1.3 nigt1.4 quadruple mutant, and a complemented line (Comp1-1.2). Fourteen-day-old 0.5× MS-grown seedlings were used in the assay.

(F) Expression levels of NIGT1.1 through 1.4 in roots in response to changes in N and P availability. Seven-day-old 0.5× MS-grown wild-type seedlings were transferred to and incubated on MGRL plates with 5 mM NH4NO3 and 1.75 mM phosphate (+N+P), without a nitrogen source (-N+P), without phosphate (+N-P), or without nitrogen and phosphate (-N-P) for 5 d.

(A) and (D) Transgenic seedlings constitutively expressing NIGT1.1-GFP (NIGT1.1ox) or NIGT1.2-GFP (NIGT1.2ox) fusion genes or GFP (GFPox) under the control of the CaMV 35S promoter were analyzed. Expression levels were analyzed by qRT-PCR and normalized using At4g34270 as an internal control.

(A) A model is proposed for NIGT1 function in the regulation of N starvation responses. Under N starvation, low NIGT1 expression allows N starvation-responsive genes such as genes involved in N uptake, recycling, remobilization, and signaling to be upregulated, leading to N starvation responses. Upon N supplementation, NIGT1s are rapidly induced and directly repress N starvation-responsive genes and attenuate N starvation responses.

(B) A model for the role of NIGT1s in balancing N starvation and Pi starvation responses (PSR). NIGT1 expression is positively regulated by Pi deficiency only under high N availability. NIGT1s have a dual role both as a direct repressor of N starvation responses and as an indirect sensitizer of Pi starvation responses by directly repressing repressors of PSR (SPX1, SPX2, SPX4, and PHO2) to balance N and Pi starvation responses.

In conclusion, we propose a mechanism that, after N supplementation and/or under high N availability conditions, accounts for the direct repression of N starvation responsive genes by NIGT1s. This optimizes N acquisition and utilization under fluctuating N availability and demand (Figure 12A). We also found that NIGT1s act to balance N and Pi starvation responses (Figure 12B). NIGT1 homologs are widely conserved among higher plants but can be phylogenetically divided into monocot and dicot types (Supplemental Figure 11). Although the role of rice NIGT1 in the regulation of N starvation responses has not been studied, rice NIGT1 shares many features with Arabidopsis NIGT1s, such as transcriptional repressor activity, binding motifs, auto-negative feedback regulation, and nitrate-inducible expression (Sawaki et al., 2013). This mechanism might be conserved among monocots and dicots and thus may be a key factor for studying N responses among all of the important crop plants.

Constructs expressing NIGT1.1-GFP or NIGT1.2-GFP translational fusion proteins under the control of their native promoters were used to observe intercellular localization of NIGT1 (Figure 2A) and in complementation tests using the multiple mutants (Supplemental Figure 5). These constructs were generated by amplifying a genomic fragment encompassing the promoter and coding region (without a stop codon) of NIGT1.1 or NIGT1.2. These amplicons were cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and integrated into the Gateway binary vector pBA002a-GFP to generate pNIGT1.1: NIGT1.1-GFP and pNIGT1.2:NIGT1.2-GFP.

To generate β-estradiol-inducible NIGT1 overexpression lines, genomic fragments encompassing the coding regions of NIGT1.1, NIGT1.2, NIGT1.3, or NIGT1.4 (without a stop codon) were amplified and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and then integrated into the Gateway binary vector pER8-derivative pER8-GW-FLAG (Zuo et al., 2000), to generate a construct that expresses NIGT1s under control of the β-estradiol-inducible promoter (eNIGT1.1ox, eNIGT1.2ox, eNIGT1.3ox, and eIGT1.4ox). To create β-estradiol-inducible NIGT1-VP overexpression lines, the full-length coding regions of NIGT1.1 or NIGT1.2 without stop codons were amplified from Arabidopsis cDNA and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and integrated into pER8-VP, a pER8-derivative, to generate constructs that express NIGT1.1 or NIGT1.2 fused with two stringent transcriptional activation domains VP16 (VP) at their C termini under control of the β-estradiol-inducible promoter (pER-NIGT1.1-VP or pER-NIGT1.2-VP).

To make constitutive NIGT1 overexpression lines, a genomic fragment encompassing the coding region of NIGT1.1 or NIGT1.2 (without a stop codon) was amplified and cloned into pENTR/D-TOPO vector (Invitrogen), sequenced, and then integrated into the Gateway binary vector pBA002-GFP (a derivative of pBA002-HA; Kiba et al., 2012), to generate constructs that express the NIGT1.1-GFP or NIGT1.2-GFP fusion genes or GFP alone under regulation of the CaMV 35S promoter (NIGT1.1ox, NIGT1.2ox, or GFPox).

GFP Imaging

For whole-plant GFP imaging, chlorophyll autofluorescence and GFP fluorescence were visualized using a FluorImager 595 (Molecular Dynamics). Chlorophyll autofluorescence and the GFP signal were obtained at 488-nm excitation using a 610RG filter and at 488-nm excitation using a 530DF30 filter.

For analysis by confocal microscopy, pNIGT1.1:NIGT1.1-GFP and pNIGT1.2:NIGT1.2-GFP seedlings grown on 0.5× MS for 10 d were observed with a Zeiss LSM700 confocal imaging system.

qRT-PCR

Total RNA was extracted from root and shoot samples using an RNeasy Plant Mini kit (Qiagen) with the RNase-Free DNase set (Qiagen). First-strand cDNA was synthesized from total RNA with the ReverTra Ace qPCR RT Master Mix (Toyobo). qRT-PCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with the KAPA SYBR Fast qPCR kit (KAPA Biosystems). At4g34270 was used as an internal control because this gene is one of the most stably expressed genes in Arabidopsis (Dekkers et al., 2012). Similar results were obtained using other internal control genes (ACT8). Primer sets are listed in Supplemental Table 2.

Y1H Screening

Y1H screening was performed using the Matchmaker Gold Yeast One-Hybrid System (Clontech) according to the manufacturer’s protocol. Three tandem copies of the P389 NRT2.4 promoter were cloned into pAbAi. The resulting plasmid was linearized with BbsI (New England Bio Labs) and transformed into the Y1H Gold strain to generate a bait-reporter yeast. This strain was then transformed with the pGAD424-based Arabidopsis transcription factor library, which contains 1498 Arabidopsis transcription factor cDNAs (Mitsuda et al., 2010). Transformants were spread on SD/-Leu medium with 100 ng/mL aureobasidin A and colonies were picked based on their growth. Plasmids isolated from each colony were retransformed to confirm positive interactions. Validated plasmids were then sequenced

Transient Assays in Arabidopsis Seedlings

Reporter, effector, and reference plasmids were codelivered to 10-d-old MS-grown Arabidopsis Col-0 seedlings by particle bombardment (-1000/He; Bio-Rad) as described previously (Nakamichi et al., 2010). Reporter and reference values were measured using the Dual-Luciferase Reporter Assay System (Promega) and Mithras LB940 (Berthold). Promoter activity was calculated as the firefly luciferase (FLUC)/Renilla luciferase (RLUC) ratio. The reporter plasmid contains FLUC downstream of the promoter of interest, and the reference plasmid harbors the ProCaMV35S:RLUC fusion gene (Fujimoto et al., 2000; Nakamichi et al., 2010). For analysis of the NIGT1-NRT2.4 promoter interaction (Figures 1E and 2D to 2G), an effector plasmid that expresses each NIGT1 family member fused with two stringent transcriptional activation domains VP16 (VP) at its C terminus under the CaMV 35S promoter was constructed as described previously (Fujimoto et al., 2000; Nakamichi et al., 2010). To construct reporter plasmids harboring a mutant NRT2.4 promoter that was mutated at the B1 motif (B1m), the B2 motifs (B2m), or in both the B1 and B2 motifs (B1mB2m), mutant promoters were synthesized (Eurofins Genomics) and cloned into the reporter plasmid. For transcriptional repressor activity assays (Figure 2B), a reporter plasmid harboring GAL4 promoter:FLUC and an effector plasmid that expresses each NIGT1 family member fused with GAL4 DNA binding domain at its N terminus under the CaMV 35S promoter were constructed as described previously (Fujimoto et al., 2000; Nakamichi et al., 2010).

ChIP Assays

Transgenic lines expressing GFP (GFPox), NIGT1.1-GFP (NIGT1.1ox), or NIGT1.2-GFP (NIGT1.2ox) fusion genes under control of the CaMV 35S promoter were used for immunoprecipitation with an anti-GFP antibody (Abcam, ab290, lot GR186433-3). Transgenic lines expressing GFP-FLAG (eGFPox), NIGT1.3-FLAG (eNIGT1.3ox), or NIGT1.4-FLAG (eNIGT1.4ox) fusion genes under control of the β-estradiol-inducible promoter treated with 10 μM β-estradiol for 24 h were used for immunoprecipitation with an anti-FLAG antibody (MBL Life Science, M185, lot 011).

Two grams of seedlings were harvested and cross-linked in 40 mL of 1% formaldehyde under vacuum for 20 min at room temperature. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min under vacuum. The cross-linked sample was frozen in liquid N and ground to a powder with a mortar and pestle in liquid N. The powder was added to 30 mL of ice-cold extraction buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 5 mM 2-mercaptoethanol, and complete protease inhibitor cocktail tablets [Roche]), and the mixture was homogenized with a Polytron (Kinematica) at 10,000 rpm for 2 min. After filtration through two layers of Miracloth (Millipore), the solution was centrifuged at 3000g at 4°C for 20 min. The pellet was suspended in 1 mL of resuspension buffer 1 (0.25 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 5 mM 2-mercaptoethanol, 1% Triton X-100, and complete protease inhibitor cocktail tablets). The resuspension was centrifuged at 12,000g at 4°C for 10 min and the pellet was suspended in 300 μL resuspension buffer 2 (1.7 M sucrose, 10 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 5 mM 2-mercaptoethanol, 0.15% Triton X-100, and complete protease inhibitor cocktail tablets [Roche]). The resuspension was layered over 300 μL of resuspension buffer 2 in a 1.5 mL microcentrifuge tube and centrifuged at 16,000g at 4°C for 60 min. The pellet was resuspended in 300 μL of sonication buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, and complete protease inhibitor cocktail tablets), sonicated five times for 15 s at setting 3 (TAITEC VP30S), and then centrifuged at 12,000g at 4°C for 5 min. The supernatant was recovered as a chromatin complex solution, and 10 μL of the solution was stored as the input fraction. The chromatin complex solution was diluted 10-fold with ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.0, 1.2 mM EDTA, 0.2% Triton X-100, and 167 mM NaCl). Forty microliters of Dynabeads protein G (Thermo Fisher Scientific) was added to preclear the solution during an incubation for 1 h at 4°C. Then, 5 μL of anti-GFP antibody or FLAG antibody was added to 1 mL of precleared solution (1/200 dilution) and incubated overnight at 4°C. This was followed by incubation with 40 μL of Dynabeads protein G (Thermo Fisher Scientific). The beads were washed three times with 1 mL of low-salt buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, and 167 mM NaCl) and then three times with high-salt buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, and 400 mM NaCl) three times each. The immunocomplex was eluted with 100 μL of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS) at 65°C for 15 min. Eluent and input fractions were incubated at 65°C overnight followed by digestion with 5 μL Proteinase K (Takara) for 1 h at 37°C to reverse cross-linking and to remove proteins. DNA was purified with a QIAquick PCR purification kit (Qiagen), and the amount precipitated was determined by qPCR using the primer sets listed in Supplemental Table 2. qPCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems) with a KAPA SYBR Fast qPCR kit (KAPA Biosystems). The enrichment value (%input) of each fragment was calculated by normalizing the amount of fragment in the immunoprecipitate against the amount in the input.

Microarray Analysis

Total RNA was extracted using an RNeasy Plant Mini kit with the RNase-Free DNase kit (Qiagen) from shoots and roots of wild-type Col-0, NIGT1.2ox line 1, and pER-NIGT1.2-VP line 2 plants (n = 3 independent pools of indicated tissues from more than 10 plants). Col-0 seedlings grown in 0.5× MS agar plates for 7 d were transferred to and incubated in MGRL plates with 5 mM NH4NO3 (WT_Nsuf) or without nitrogen source (WT_Nstv) for 3 d. NIGT1.2ox line1 seedlings grown in 0.5× MS agar plates for 7 d were transferred to and incubated in MGRL plates without a nitrogen source (OX_Nstv) for 3 d. pER-NIGT1.2-VP line 2 seedlings grown in MGRL plates containing 5 mM NH4NO3 for 9 d were transferred to and incubated in MGRL plates containing 5 mM NH4NO3 (VP_Mock) or 5 mM NH4NO3 and 10 μM β-estradiol (VP_+Est) for 24 h. Three biological replicates were analyzed with the Affymetrix Arabidopsis ATH1 Genome Array. RNA quality was determined on a Bioanalyzer (Agilent), and RNA amplification, labeling, and hybridization were conducted with the 3′ IVT Express Kit (Affymetrix) according to the manufacturer’s instructions. Processing and analyses of microarray data were performed using R software (version 3.2.3 or later; https://www.r-project.org/). Gene expression levels, represented by signal intensities of corresponding probes, were obtained as logarithms to base 2 after normalization among the 15 microarray chips by the robust multiarray average method applying the “affy” library (Gautier et al., 2004). After normalization, probes annotated as “control sequence” were excluded from the following analyses. The 22,746 probes annotated as “exemplar sequence” were applied. Probes showing statistically different signal intensities between conditions or strains were determined with a two-tailed Welch’s t test (P < 0.05) (Welch, 1947) following a multiple testing correction by the Benjamini-Hochberg procedure (FDR < 0.1) (Benjamini and Hochberg, 1995). Overrepresented GO terms were determined by Fisher’s exact test (Fisher, 1922) followed by the Benjamini-Hochberg procedure (FDR < 0.05). For this analysis, probes were annotated with the GO terms by referring probe-AGI code correspondence provided by the manufacturer and the GO Slim classification downloaded from the TAIR database (https://www.arabidopsis.org) as of April 21, 2016.

Chlorophyll, Soluble Protein, and Total Amino Acid Measurements

Chlorophyll measurements were performed as described previously (Porra et al., 1989). Briefly, chlorophyll was extracted with N,N-dimethylformamide for 24 h at 4°C in the dark followed by measurement of absorbance at 663.8 nm (for chlorophyll a) and 646.8 nm (for chlorophyll b) in a spectrophotometer (UV-1650PC; Shimadzu).

Determination of soluble protein content was conducted as previously described (Makino et al., 1985). Shoot and root samples were homogenized and suspended in 50 mM Na-phosphate buffer (pH 7.5) containing 5 mM dithiothreitol and 12.5% (v/v) glycerol. The homogenate was centrifuged at 12,000g at 4°C for 10 min to clear cellular debris. Soluble protein in the supernatant was measured using Bio-Rad Bradford Protein Assay according to the manufacturer’s instructions.

For determination of free amino acids, root samples were powdered in liquid nitrogen, mixed with 10 volumes of 10 mM HCl containing 0.2 mM methionine sulfone as an internal control, and centrifuged at 18,000g at 4°C for 5 min, and the supernatant was filtered through an Ultrafree-MC filter (0.22 μm; Millipore). The filtrate was derivatized using a derivatization regent (a mixture of methanol:water:triethylamine:phenylisothiocyanate at the volume ratio of 7:1:1:1). Derivatized amino acid levels were determined using a high performance liquid chromatography system (Alliance 2695; Waters) linked to a photodiode array detector (2995; Waters) with Pico-Tag eluents (Waters) and a PicoTag reverse phase column (3.9 mm × 300 mm; Waters) according to the manufacturer’s instructions.

Nitrogen Content and Root 15N Influx

Influx of 15NO3 was assayed as previously described (Lezhneva et al., 2014) with some modifications. Seedling roots were submerged in 0.1 mM CaSO4 for 1 min and then transferred to complete nutrient solution containing 15NO3 (atom% 15N: 99%) at the indicated concentrations for 5 to 15 min. Roots were transferred to 0.1 mM CaSO4 for 1 min before harvest. Whole seedlings were dried completely at 80°C and analyzed using a FlashEA1112 elemental analyzer (Thermo) and DELTA plus Advantage Isotope-ratio mass spectrometer (Themo) coupled via a ConFlo III interface (Thermo). Influx of 15NO3- was calculated from the total N and 15N content in whole seedlings.

Nitrate and Phosphate Measurements

Whole seedlings, shoots, or roots were weighed and collected in 2.0 mL Eppendorf Safe-Lock tubes (Eppendorf). Samples were pulverized in liquid N, and 1 mL of milli-Q water was added to each tube to extract nitrate and free phosphate (Pi). After centrifugation, supernatant nitrate concentrations were quantified by a high performance liquid chromatography system (alliance 2695; Waters) linked to a photodiode array detector (2995; Waters) using an anion exchange column (Waters Spherisorb S5 SAX; 50 mM K2HPO4/KH2PO4, pH 3.7 at a rate of 1 mL/min) (Kiba et al., 2012) or by a spectrophotometric method as previously described (Miranda et al., 2001). Similar results were obtained by both methods. Phosphate levels were measured using the Malachite Green Phosphate Assay Kit (POMG-25H; BioAssay Systems).

Statistical Analysis

Data are given as means ± sd of one representative experiment. In order to examine whether values such as promoter activity, gene expression, and nitrate influx were significantly different between treatments or genotypes, Student’s t tests and Tukey’s honestly significant difference (HSD) tests were performed using KaleidaGraph version 4.1 software (Synergy Software).

Acknowledgments

We thank the Arabidopsis Biological Resource Center and Nottingham Arabidopsis Stock Centre for supplying T-DNA insertion lines. This work was supported in part by the Japan Society for the Promotion of Science KAKENHI Grants 26712009 and 17K07705 to T. Kiba., 25252014 and 26221103 to S.Y., and 15H05616 to M.K., and by JST CREST Grant JPMJCR15O5 to S.Y.

Footnotes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Takatoshi Kiba (takatoshi.kiba{at}riken.jp).

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